Rhizopus homothallicus, an emerging pathogen causing cavitary lung lesions

Introduction. Rhizopus homothallicus is an emerging pathogen that causes pulmonary mucormycosis. Case Presentation. We report a case of pneumonia caused by R. homothallicus in a 54-year-old type 2 diabetic patient. The organism was isolated from bronchoalveolar lavage fluid and preliminarily identified by fungal morphology and finally by sequencing of the internal transcribed spacer region. Conclusion. Mucormycosis may be associated with cavitary lung lesions against a backdrop of poorly controlled diabetes or other immunosuppressed states. Pulmonary mucormycosis may have variable clinical and radiological presentations. Therefore, strong clinical suspicion and prompt management can address the high fatality associated with the disease.


INTRODUCTION
Ubiquitous fungi such as Rhizopus, Absidia, Rhizomucor, Mucor and Cunninghamella, species of the order Mucorales, are found in the soil and cause opportunistic infections. The known predisposing factors in patients with mucormycosis are uncontrolled diabetes, neutropenia, carcinomas, immunosuppressive therapy, deferoxamine therapy and voriconazole prophylaxis [1,2]. Among the genera Rhizopus, Rhizopus homothallicus is being reported in a large number of cases and is an emerging pathogen [1]. The sporangiospores released by Mucorales gain entry to the upper or lower airways through aerosolization. Pulmonary mucormycosis is second in frequency after rhino-orbital-cerebral among the reported cases of mucormycosis.

CASE PRESENTATION
A 54-year-old male with type 2 diabetes mellitus presented with complaints of intermittent fever, cough with expectoration, occasional haemoptysis and right-sided chest pain for the previous 20 days. There was no history of coronavirus disease 2019  infection in the recent past. The patient tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by RT-PCR and gave a history of vaccination with two doses of Covishield. The patient was a known case of type 2 diabetes mellitus for the previous 20 years and was on irregular medication of metformin and pioglitazone. On examination, the patient was afebrile, conscious and oriented. On auscultation, breath sounds were markedly reduced on the right side with fine crepitations. Renal function, liver function tests and urinalysis were unremarkable except for blood sugar levels of 516 mg dl −1 and total leucocyte count of 13 000 mm −3 on admission. Testing for HbA1c level was not performed. There was no evidence of acid-fast bacilli in the sputum. A serum galactomannan test was negative. Chest X-ray revealed consolidation in the middle and lower zone of the right lung (Fig. 1a). On day 4 blood parameters showed a markedly elevated total leucocyte count (>35 000 mm −3 ) with neutrophilia (>90 %) and lymphocytopenia (~3 %), and high procalcitonin (15.3 ng ml −1 ), d-dimer (10 000 ng ml −1 ), creatinine (4 mg dl −1 ), urea (200 mg dl −1 ), SGOT (209 U l −1 ) and SGPT (167 U l −1 ) levels. The patient also showed traces of ketones in urine and was subsequently shifted to the intensive care unit for close monitoring.
On admission the patient was initiated on amoxicillin/clavulanic acid, and clarithromycin. High-resolution computed tomography (HRCT) chest performed on day 2 showed a large area of consolidation with bronchiectasis, fibrosis and secondary cavitary changes showing air-fluid levels in the right lower lobe with intra-and interlobular septal thickening with ground glass haze giving a crazy paving pattern in the right lower lobes and a few patchy areas of consolidation in the right upper and middle lobe with minimal right pleural effusion (Fig. 1a). On day 4, as the patient deteriorated clinically with persistent fever, high blood counts and increasing consolidations seen on chest radiograph, the antibiotics were escalated to meropenem and teicoplanin. On day 5 due to further deterioration in the condition of the patient, fungal aetiology was suspected. A bronchoalveolar lavage sample was collected and sent for microbiological analysis. KOH examination was performed, which showed broad aseptate fungal hyphae (Fig. 1b). On day 5, the patient underwent sudden respiratory distress, for which he was intubated. Taking into account the reduced urine output and altered renal function, the patient was initiated on dialysis. The patient was started on posaconazole as the salvage therapy. On admission day 6, the patient died due to sudden cardiopulmonary arrest and could not be revived.
Fungal culture results showed a fast-growing, cottony white colony turning brown on Sabouraud dextrose agar (SDA) incubated at 25 °C and 37 °C, within 48 h of incubation. The isolate was identified as R. homothallicus based on the characteristic goldenbrown spiny zygospores and suspensor cells, along with broad, aseptate hyphae, seen on a lactophenol cotton blue (LPCB) mount (Fig. 1c). DNA was extracted and outsourced for PCR targeting the internal transcribed spacer 2 followed by Sanger sequencing. The sequence has been submitted to GenBank with accession number OP019829. A sequence alignment was performed using the ISHAM database and it was confirmed to be R. homothallicus with a similarity index of 96.97 %. The evolutionary tree was prepared using the neighbour-joining method and evolutionary analysis was conducted using mega X (Fig. 2) [3].

DISCUSSION
Rhizopus species are the most commonly isolated fungi from the patients of mucormycosis. Apophysomyces elegans, Apophysomyces variabilis and Rhizopus homothallicus are reported to be emerging species [4]. Chakrabarti et al. first reported infections due to R. homothallicus in pulmonary infection patients [5]. There are published reports of several cases that indicate the geographical niche of the fungi causing pulmonary, cutaneous and rhino orbitocerebral mucormycosis [5][6][7][8][9][10]. The incidence of the disease is high in India and also rising globally with the rise in cases of diabetes mellitus [11]. Our patient was diagnosed with sepsis and DKA and was at high risk of developing mucormycosis. In diabetic ketoacidosis (DKA), hyperglycaemia and low pH make phagocytes dysfunctional, with impaired chemotaxis and defective intracellular killing of the pathogen [12]. Mucorales possess the unique ability to acquire iron from the host, which enables the pathogens to grow. In DKA patients there is proton-mediated release of ferric ions from transferrin [12]. Another mechanism has been identified through which there is DKA-enhanced upregulation of glucose-regulated protein (GRP78), which acts as a receptor that mediates entry and damage of endothelial cells by Mucorales [13]. The growth of the Rhizopus in the body causes extensive vessel thrombosis and tissue necrosis, as a result of which the fungus disseminates to other organs [12]. Pulmonary mucormycosis has been described in patients with associated immunosuppression, such as neutropenia or graftversus-host disease, whereas rhino-orbital disease is typically reported in patients with uncontrolled diabetes [14]. The patient in the present setting had longstanding poorly controlled diabetes and presented with DKA. Radiology is considered to be a sensitive marker of pulmonary fungal infection, and the presence of the reverse halo sign on computed tomography (CT) scans has been suggested to be a strong indicator of pulmonary mucormycosis [15]. The present case presented with cavitary pneumonia, which is a rare finding [5]. The clinical findings and chest imaging features are not specific, hence pulmonary mucormycosis is easily misdiagnosed, which can result in serious consequences.
Microscopy and culture of the bronchoalveolar lavage fluid remain the gold standard to establish a diagnosis of pulmonary mucormycosis. The first-line drug is liposomal amphotericin B; however, the clinical team, on assessing the risk/benefit in view of the nephrotoxicity of the drug, initiated posaconazole. The patient succumbed to the disease due to delay in reaching to the healthcare facility and also a delay in initiating antifungal treatment, as fungal infection was not suspected at the outset. Early surgical treatment, appropriate antifungal therapy, and control of predisposing factors are of great importance in the treatment of such cases [16]. However, as patients with pulmonary mucormycosis deteriorate rapidly, the overall mortality rate reported is 40-76 % [17].
The internal transcribed spacer sequence showed maximum similarity to sequences KU926333 and MK351861, as shown in Fig. 2. MK351861 was isolated from soil at Ratapani wildlife sanctuary in Raisen, Madhya Pradesh [18]. Sequence KU926333 was isolated from the lung biopsy sample of a fatal case of pulmonary mucormycosis in a patient with unchecked diabetes mellitus from Paris [19]. Genomic identification of the fungi is strongly recommended for improved epidemiological understanding of mucormycosis.

Funding information
This work received no specific grant from any funding agency.

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Peer review history 1. Provide further information on the duration of the diabetes? what was the diabetes treatment the patient was on if any did you get information on HbA1c?.
The patient was a known case of T2DM for 20 years. The patient was on irregular medication of metformin and pioglitazone. Testing for HbA1c level was not done. Random blood sugar at time of admission was 516 mg/dL. patient also showed traces of ketones in urine. Subsequently patient was initiated on insulin infusion and 2 hourly monitoring of blood sugar levels was being done.
Added line 61-62 Added line 67-68 2. Also, it would be great to know not only the total WBC count but also the type of WBC increased (neutrophils?) and also to know if you found some other inflammatory markers increased. 1. On admission the patient was initiated on amoxyclav, and clarithromycin which was subsequently revised to meropenem and teicoplanin 2. The first line drug is liposomal amphotericin B, however, the clinical team on assessing therisk/benefit ratio of associated nephrotoxicity of the drug initiated posaconazole.
Added line 75, 83 Added line 148-149 5. Finally, I think you need to add more details about the molecular identification, your document says you extracted DNA followed by Sanger sequencing, but if I'm not mistaken you amplified the DNA ITS2 region before sequencing, this need to be said and also mentioning the primers used if you designed them or if you used previously used primers and add the reference, same as details on the PCR procedure.
DNA was extracted and outsourced PCR was performed targeting the ITS-2 region and the products were further used for Sanger's sequencing. The consensus sequence generated was and submitted to GenBank (Accession No. OP019829) Added Line 95 6. I would highly recommend figures description have more information about the findings and also clearly identify these findings using marks/ arrows to identify them in the figure.
Done Figure Legend with detailed description of the scans is incorporated

Reviewer #2
Please include a histopathology photo of the BAL sample in KOH examination which showed broad aseptate fungal hyphae, in current paper Added Figure 1B Reviewer #3:The description of the case is acceptable. However, the discussion needs major improvement. The importance of the case-report lies in its striking clinical findings. Also, the discussion should be able to highlight your findings and provide extra information about the organism to the readers. The first line drug is liposomal amphotericin B, however, the clinical team on assessing the risk/benefit ratio of associated nephrotoxicity of the drug initiated posaconazole.
Added line 148-149 6. Line 102: Please mention the region/area of the world from which these cases are isolates.
The isolate from Paris was obtained from lung biopsy of a fatal case of pulmonary mucormycosis while isolate from India was obtained from soil.
Added line 156-157 7. Discussion on whole should be rewritten: a. Firstly, the discussion should include the importance of R. homothallicuswith its prevalence in the world. b. Secondly, the authors should discuss the pathogenesis of mucormycosis and how this patient was susceptible to mucormycosis c. Thirdly, Control of hyperglycaemia, early treatment with liposomal amphotericin B, and surgery are essential for the successful management of mucormycosis. The authors should discuss these points and what was done and what could not be done in their patient. d. Lastly, discuss the causes of mortality in pulmonary mucormycosis. Also the trend of case fatality.